During inflammation, epithelial cells of the intestine are exposed to proteases from inflammatory cells (e.g. mast cell tryptase), the epithelium (extrapancreatic trypsins) and the circulation (coagulation factors). However, nothing is known about the biological actions of these proteases or the mechanisms of their effects. This proposal examines the hypothesis that proteases that are generated during inflammation signal to intestinal epithelial cells by cleaving protease-activated receptors (PARs); activation of PARs increases paracellular permeability and thereby exacerbates inflammation. This hypothesis is tested in cell-based model systems (cultured colonocytes) and intact animals. The proposal focuses on PAR2, a receptor for tryptase, trypsins and coagulation factors Vlla and Xa. Aim 1 will investigate the importance of receptor trafficking in colonocytes to the initiation and termination of PAR2 signaling. Aim 1A will examine the formation of a PAR2 "signaling module" in endosomes that determines the function of mitogen activated protein kinases and thereby plays a major role in signaling in colonocytes. Aim 1B will investigate the mechanisms that terminate PAR2 signaling. Since proteolytic activation is irreversible, efficient mechanisms must terminate PAR2 signaling. Experiments will examine the role of the E3 ubiquitin ligase c-Cbl in ubiquitination of PAR2 and will determine the importance of ubiquitination for lysosomal trafficking of PAR2. Aim 2 will investigate the mechanisms by which proteases regulate paracellular permeability of colonocytes in culture. Aim 2A will determine if proteases that are generated during inflammation can signal to colonocytes by cleaving and activating PAR2. Aim 2B will investigate the signal transduction mechanisms by which PAR2 regulates the formation of tight junctions. Aim 3 will investigate the role of proteases and PAR2 in the control of paracellular permeability and inflammation in animal models of colitis. Aim 3A will characterize the proteases that are generated in the inflamed colon and will determine if proteases increase mucosal permeability and cause inflammation by activating PAR2. Aim 3B will define the contribution of PAR2 and tryptase in colitis using PAR2 knockout animals and specific inhibitors. Thus, aim 1 will provide basic information about mechanisms of signal transduction by G-protein coupled receptors, and aims 2 and 3 identify novel mechanisms by which proteases and their receptors regulate epithelial permeability and mucosal inflammation.